Proteasomal down-regulation of the proapoptotic MST2 pathway contributes to BRAF inhibitor resistance in melanoma.

The RAS-RAF-MEK-ERK pathway is hyperactivated in most malignant melanomas, and mutations in BRAF or NRAS account for most of these cases. BRAF inhibitors (BRAFi) are highly efficient for treating patients with BRAFV600E mutations, but tumours frequently acquire resistance within a few months. Multiple resistance mechanisms have been identified, due to mutations or network adaptations that revive ERK signalling. We have previously shown that RAF proteins inhibit the MST2 proapoptotic pathway in a kinase-independent fashion. Here, we have investigated the role of the MST2 pathway in mediating resistance to BRAFi. We show that the BRAFV600E mutant protein, but not the wild-type BRAF protein, binds to MST2 inhibiting its proapoptotic signalling. Down-regulation of MST2 reduces BRAFi-induced apoptosis. In BRAFi-resistant cell lines, MST2 pathway proteins are down-regulated by ubiquitination and subsequent proteasomal degradation rendering cells refractory to MST2 pathway-induced apoptosis. Restoration of apoptosis can be achieved by increasing MST2 pathway protein expression using proteasome inhibitors. In summary, we show that the MST2 pathway plays a role in the acquisition of BRAFi resistance in melanoma.

Hypercoagulable Disorders in Orthopaedics: Etiology, Considerations, and Management.

¼: Hypercoagulable disorders (HCDs) can be inherited or acquired. An HCD of either etiology increases the chance of venous thromboembolic events (VTEs). ¼: Patients with an HCD often have the condition discovered only after surgical complications. ¼: We recommend that patients with a concern for or a known HCD be referred to the appropriate hematological specialist for workup and treatment. ¼: Tourniquet use in the orthopaedic patient with an HCD is understudied and controversial. We recommend that tourniquets be avoided in the surgical management of patients with an HCD, if possible. When tourniquets are applied to patients with unknown HCD status, close follow-up and vigilant postoperative examinations should be undertaken.

Comparison of 3 C-Arm Draping Techniques to Prevent Far Side Contamination.

OBJECTIVE: To assess the effectiveness of reducing contamination using 2 methods of C-Arm draping compared with traditional methods. MATERIALS AND METHODS: The authors simulated an operating room using an extremity drape, commercially available C-Arm drapes, and C-Arm. A black light was placed above the field. A fluorescent powder was placed on the nonsterile portions of the field. Baseline light intensity was recorded by photo. The C-Arm was brought into the surgical field for orthogonal imaging for 15 cycles. A repeat photograph was taken to measure the increase in intensity of the fluorescent powder to assess degree of contamination. This was repeated 5 times for each configuration: standard C-Arm drape, a proprietary close-fitting drape, and a split drape secured to the far side with the split wrapped around the C-Arm receiver. Light intensity difference was measured and average change in intensity was compared. RESULTS: Compared with standard draping, the proprietary close-fitting drape resulted in a 71.3% decrease in contamination (4.84% vs. 16.90%, P = 0.101) that trended toward significance and the split drape resulted in a 99.5% decrease (0.09% vs. 16.90%, P = 0.017) that was statistically significant. CONCLUSION: Far side contamination can be reduced by using a split drape connecting the operative table to the C-Arm receiver, effectively "sealing off" contaminants. The proprietary close-fitting drape may also decrease contamination, but this was not statistically significant in this study. Use of the split drape technique will help prevent contamination and may ultimately lead to decreased infection risk.

Kinome rewiring during acquired drug resistance in neuroendocrine neoplasms.

Although there is evidence of a significant rise of neuroendocrine neoplasms (NENs) incidence, current treatments are largely insufficient due to somewhat poor knowledge of these tumours. Despite showing differentiated features, NENs exhibit therapeutic resistance to most common treatments, similar to other cancers in many instances. Molecular mechanisms responsible for this resistance phenomenon are badly understood. We aimed at identifying signalling partners responsible of acquired resistance to treatments in order to develop novel therapeutic strategies. We engineered QGP-1 cells resistant to current leading treatments, the chemotherapeutic agent oxaliplatin and the mTor inhibitor everolimus. Cells were chronically exposed to the drugs and assessed for acquired resistance by viability assay. We used microarray-based kinomics to obtain highthroughput kinase activity profiles from drug sensitive vs resistant cells and identified 'hit' kinases hyperactivated in drug-resistant cells, including kinases from FGFR family, cyclin-dependant kinases and PKCs in oxaliplatin-resistant (R-Ox) QGP-1 cells. We then validated these 'hit' kinases and observed that ERK signalling is specifically enhanced in QGP-1 R-Ox cells. Finally, we assessed drug-resistant cells sensitivity to pharmacological inhibition of 'hit' kinases or their signalling partners. We found that FGFR inhibition markedly decreased ERK signalling and cell viability in QGP-1 R-Ox cells. These results suggest that the FGFR/ERK axis is hyperactivated in response to oxaliplatin-based chemotherapeutic strategy. Thus, this sensitive approach, based on the study of kinome activity, allows identifying potential candidates involved in drug resistance in NENs and may be used to broadly investigate markers of NENs therapeutic response.

Relevance of neuroendocrine tumours models assessed by kinomic profiling.

Although there is evidence of a significant rise of neuroendocrine tumours (NETs) incidence, current treatments are largely insufficient due to somewhat poor knowledge of these tumours. Despite many efforts achieved to expose driver oncogene mutations in NETs, the genetic landscape of NETs is characterized by relatively few mutations and chromosomal aberrations per tumour compared with other tumour types. In addition, NETs display few actionable mutations providing compelling rationale for targeted therapies. Recent works aiming at characterizing currently used NETs in vitro models at the genomic level raised concerns on their reliability as bona fide tools to study NETs biology. However, the lack of actionable mutation in NETs implies that sole use of genomic is not sufficient to describe these models and establish appropriate therapeutic strategies. Several kinases and kinase-involving signalling pathways have been demonstrated as abnormally regulated in NETs. Yet, kinases have only been investigated regardless of their involvement in large intracellular signalling networks. In order to assess the validity of in vitro NETs models to study NETs biology, "next-generation" high throughput functional technologies based on "kinome-wide activity" will demonstrate the similarities between signalling pathways in NETs models and patients' samples. These approaches will significantly assist in identifying actionable alterations in NETs signalling pathways and guide patient stratification into early-phase clinical trials based on kinase inhibition targeted therapies.

Longitudinal identification of clinically distinct neurophenotypes in young children with fragile X syndrome.

Fragile X syndrome (FXS), due to mutations of the FMR1 gene, is the most common known inherited cause of developmental disability. The cognitive, behavioral, and neurological phenotypes observed in affected individuals can vary considerably, making it difficult to predict outcomes and determine the need for interventions. We sought to examine early structural brain growth as a potential marker for identification of clinically meaningful subgroups. Participants included 42 very young boys with FXS who completed a T1-weighted anatomical MRI and cognitive/behavioral assessment at two longitudinal time points, with mean ages of 2.89 y and 4.91 y. Topological data analysis (TDA), an unsupervised approach to multivariate pattern analysis, was applied to the longitudinal anatomical data to identify coherent but heretofore unknown subgroups. TDA revealed two large subgroups within the study population based solely on longitudinal MRI data. Post hoc comparisons of cognition, adaptive functioning, and autism severity scores between these groups demonstrated that one group was consistently higher functioning on all measures at both time points, with pronounced and significant unidirectional differences (P < 0.05 for time point 1 and/or time point 2 for each measure). These results support the existence of two longitudinally defined, neuroanatomically distinct, and clinically relevant phenotypes among boys with FXS. If confirmed by additional analyses, such information may be used to predict outcomes and guide design of targeted therapies. Furthermore, TDA of longitudinal anatomical MRI data may represent a useful method for reliably and objectively defining subtypes within other neuropsychiatric disorders.

Pasireotide is more effective than octreotide, alone or combined with everolimus on human meningioma in vitro.

Pasireotide is a somatostatin analog (SSA) that targets somatostatin receptor subtype 1 (SST1), SST2, SST3, and SST5 with a high affinity. Pasireotide has a better antisecretory effect in acromegaly, Cushing's disease, and neuroendocrine tumors than octreotide. In this study, we compared the effects of pasireotide to those of octreotide in vitro on meningioma primary cell cultures, both alone and in combination with the mTOR inhibitor everolimus. Significant mRNA expression levels of SST1, SST2, and SST5 were observed in 40.5%, 100%, and 35% of meningioma samples, respectively. Pasireotide had a significantly stronger inhibitory effect on cell proliferation than octreotide. The effect of pasireotide, but not of octreotide, was significantly stronger in the group expressing the highest level of SST1 mRNA. Combined treatment with pasireotide and everolimus induced a higher reduction in cell viability than that with octreotide plus everolimus. Moreover, pasireotide decreased Akt phosphorylation and reversed everolimus-induced Akt hyperphosphorylation to a higher degree than octreotide. Using 4E-BP1 siRNA (si4E-BP), we demonstrated that 4E-BP1 protein silencing significantly reversed the response to everolimus, both alone and in combination with SSAs. Moreover, si4E-BP completely reversed the inhibition of cyclin D1 expression level and the increase in p27kip1 induced by SSAs, both alone and in combination with everolimus. Our results strongly support the need for further studies on the combination of pasireotide and everolimus in medical therapy for meningiomas.

Anti-proliferative and anti-secretory effects of everolimus on human pancreatic neuroendocrine tumors primary cultures: is there any benefit from combination with somatostatin analogs?

Therapeutic management of gastroenteropancreatic neuroendocrine tumors (GEP-NETs) is challenging. The mammalian target of rapamycin (mTOR) inhibitor everolimus recently obtained approval from the Food and Drug Administration for the treatment of patients with advanced pancreatic neuroendocrine tumors (pNETs). Despite its promising antitumor efficacy observed in cell lines, clinical benefit for patients is unsatisfactory. The limited therapeutic potential of everolimus in cancer cells has been attributed to Akt activation due to feedback loops relief following mTOR inhibition. Combined inhibition of Akt might then improve everolimus antitumoral effect. In this regard, the somatostatin analog (SSA) octreotide has been shown to repress the PI3K/Akt pathway in some tumor cell lines. Moreover, SSAs are well tolerated and routinely used to reduce symptoms caused by peptide release in patients carrying functional GEP-NETs. We have recently established and characterized primary cultures of human pNETs and demonstrated the anti-proliferative effects of both octreotide and pasireotide. In this study, we aim at determining the antitumor efficacy of everolimus alone or in combination with the SSAs octreotide and pasireotide in primary cultures of pNETs. Everolimus reduced both Chromogranin A secretion and cell viability and upregulated Akt activity in single treatment. Its anti-proliferative and anti-secretory efficacy was not improved combined with the SSAs. Both SSAs did not overcome everolimus-induced Akt upregulation. Furthermore, caspase-dependent apoptosis induced by SSAs was lost in combined treatments. These molecular events provide the first evidence supporting the lack of marked benefit in patients co-treated with everolimus and SSA.

Octreotide therapy in meningiomas: in vitro study, clinical correlation, and literature review.

OBJECTIVE Meningiomas express somatostatin receptor subtype 2 (SST2), which is targeted by the somatostatin analog octreotide. However, to date, using somatostatin analog therapy for the treatment of these tumors in clinical practice has been debated. This study aims to clarify the in vitro effects of octreotide on meningiomas for precise clinical applications. METHODS The effects of octreotide were analyzed in a large series of 80 meningiomas, including 31 World Health Organization (WHO) Grade II and 4 WHO Grade III tumors, using fresh primary cell cultures to study the impact on cell viability, apoptosis, and signal transduction pathways. RESULTS SST2 mRNA was detected in 100% of the tested meningiomas at levels similar to those observed in other SST2-expressing tumors, neuroendocrine tumors, or pituitary adenomas. Octreotide significantly decreased cell proliferation in 88% of meningiomas but did not induce cell death. On average, cell proliferation was more inhibited in the meningioma group expressing a high level of SST2 than in the low-SST2 group. Moreover, octreotide response was positively correlated to the level of merlin protein and inversely correlated to the level of phosphorylated p70-S6 kinase, a downstream effector of the PI3K/Akt/mammalian target of rapamycin (mTOR) pathway. Octreotide inhibited Akt phosphorylation and activated tyrosine phosphatase without impacting the extracellular regulated kinase (ERK) pathway. CONCLUSIONS Octreotide acts exclusively as an antiproliferative agent and does not promote apoptosis in meningioma in vitro. Therefore, in vivo, octreotide is likely to limit tumor growth rather than induce tumor shrinkage. A meta-analysis of the literature reveals an interest in octreotide for the treatment of WHO Grade I tumors, particularly those in the skull base for which the 6-month progression-free survival level reached 92%. Moreover, somatostatin analogs, which are well-tolerated drugs, could be of interest for use as co-targeting therapies for aggressive meningiomas.

SARAH Domain-Mediated MST2-RASSF Dimeric Interactions.

RASSF enzymes act as key apoptosis activators and tumor suppressors, being downregulated in many human cancers, although their exact regulatory roles remain unknown. A key downstream event in the RASSF pathway is the regulation of MST kinases, which are main effectors of RASSF-induced apoptosis. The regulation of MST1/2 includes both homo- and heterodimerization, mediated by helical SARAH domains, though the underlying molecular interaction mechanism is unclear. Here, we study the interactions between RASSF1A, RASSF5, and MST2 SARAH domains by using both atomistic molecular simulation techniques and experiments. We construct and study models of MST2 homodimers and MST2-RASSF SARAH heterodimers, and we identify the factors that control their high molecular stability. In addition, we also analyze both computationally and experimentally the interactions of MST2 SARAH domains with a series of synthetic peptides particularly designed to bind to it, and hope that our approach can be used to address some of the challenging problems in designing new anti-cancer drugs.

Autophosphorylation on S614 inhibits the activity and the transforming potential of BRAF.

The BRAF proto-oncogene serine/threonine-protein kinase, known as BRAF, belongs to the RAF kinase family. It regulates the MAPK/ERK signalling pathway affecting several cellular processes such as growth, survival, differentiation, and cellular transformation. BRAF is mutated in ~8% of all human cancers with the V600E mutation constituting ~90% of mutations. Here, we have used quantitative mass spectrometry to map and compare phosphorylation site patterns between BRAF and BRAF V600E. We identified sites that are shared as well as several quantitative differences in phosphorylation abundance. The highest difference is phosphorylation of S614 in the activation loop which is ~5fold enhanced in BRAF V600E. Mutation of S614 increases the kinase activity of both BRAF and BRAF V600E and the transforming ability of BRAF V600E. The phosphorylation of S614 is mitogen inducible and the result of autophosphorylation. These data suggest that phosphorylation at this site is inhibitory, and part of the physiological shut-down mechanism of BRAF signalling.

Surgical Management of Symptomatic Extensor Digitorum Brevis Manus: A Proposed Algorithm for Treatment.

First described in 1734, the extensor digitorum brevis manus (EDBM) is an anomalous extensor muscle found in the dorsum of the wrist and hand. Extensor muscle variants of the hand are not uncommon, and EDBM has an estimated reported incidence of approximately 2%. Although few extensor muscle variants become clinically significant, there is a paucity of literature discussing these anatomic variants, with most reports arising from cadaveric studies or isolated case series. Similarly, there are few established indications for surgical treatment of EDBM. In this case report, we describe the successful treatment of a young patient with persistently symptomatic anomalous extensor tendon with surgical excision and propose an algorithm for management after failure of conservative measures.

Mitogen-Inducible Gene-6 Mediates Feedback Inhibition from Mutated BRAF towards the Epidermal Growth Factor Receptor and Thereby Limits Malignant Transformation.

BRAF functions in the RAS-extracellular signal-regulated kinase (ERK) signaling cascade. Activation of this pathway is necessary to mediate the transforming potential of oncogenic BRAF, however, it may also cause a negative feedback that inhibits the epidermal growth factor receptor (EGFR). Mitogen-inducible gene-6 (MIG-6) is a potent inhibitor of the EGFR and has been demonstrated to function as a tumor suppressor. As MIG-6 can be induced via RAS-ERK signaling, we investigated its potential involvement in this negative regulatory loop. Focus formation assays were performed and demonstrated that MIG-6 significantly reduces malignant transformation induced by oncogenic BRAF. Although this genetic interaction was mirrored by a physical interaction between MIG-6 and BRAF, we did not observe a direct regulation of BRAF kinase activity by MIG-6. Interestingly, a selective chemical EGFR inhibitor suppressed transformation to a similar degree as MIG-6, whereas combining these approaches had no synergistic effect. By analyzing a range of BRAF mutated and wildtype cell line models, we could show that BRAF V600E causes a strong upregulation of MIG-6, which was mediated at the transcriptional level via the RAS-ERK pathway and resulted in downregulation of EGFR activation. This feedback loop is operational in tumors, as shown by the analysis of almost 400 patients with papillary thyroid cancer (PTC). Presence of BRAF V600E correlated with increased MIG-6 expression on the one hand, and with inactivation of the EGFR and of PI3K/AKT signaling on the other hand. Importantly, we also observed a more aggressive disease phenotype when BRAF V600E coexisted with low MIG-6 expression. Finally, analysis of methylation data was performed and revealed that higher methylation of MIG-6 correlated to its decreased expression. Taken together, we demonstrate that MIG-6 efficiently reduces cellular transformation driven by oncogenic BRAF by orchestrating a negative feedback circuit directed towards the EGFR.

Occupational Outcomes and Return to Running Following Internal Fixation of Ankle Fractures in a High-Demand Population.

BACKGROUND: Literature evaluating surgical outcomes after ankle fixation in an active patient population is limited. This study determined occupational outcomes and return to running following ankle fracture fixation in a military cohort. METHODS: All service members undergoing ankle fracture fixation at a single military hospital from August 2007 to August 2012 were reviewed. Univariate analysis determined the association between patient demographic information, type of fracture fixation, and the development of posttraumatic ankle arthritis and functional outcomes, including medical separation, return to running, and reoperation. Seventy-two primary ankle fracture fixation procedures were performed on patients with mean age of 29.1 years. The majority of patients were male (88%), were 25 years of age or older (61%), were of junior rank (57%), underwent unimalleolar fracture fixation (78%), and did not require syndesmotic fixation (54%). The average follow-up was 35.9 months. RESULTS: The mean time to radiographic union was 8.6 weeks. Twelve service members (17%) were medically separated from the military due to refractory pain following ankle fracture fixation with a minimum of 2-year occupational follow-up. Among military service members undergoing ankle fracture fixation, 64% returned to running. Service members with higher occupational demands had a statistical trend to return to running (odds ratio [OR] 2.49; 95% CI, 0.93-6.68). Junior enlisted rank was a risk factor for medical separation (OR 11.00; 95% CI, 1.34-90.57). Radiographic evidence of posttraumatic ankle osteoarthritis occurred in 8 (11%) service members. CONCLUSIONS: At mean 3-year follow-up, 83% of service members undergoing ankle fracture fixation remained on active duty or successfully completed their military service, while nearly two-thirds returned to occupationally required daily running. LEVEL OF EVIDENCE: Level IV, retrospective case series.

Competing to coordinate cell fate decisions: the MST2-Raf-1 signaling device.

How do biochemical signaling pathways generate biological specificity? This question is fundamental to modern biology, and its enigma has been accentuated by the discovery that most proteins in signaling networks serve multifunctional roles. An answer to this question may lie in analyzing network properties rather than individual traits of proteins in order to elucidate design principles of biochemical networks that enable biological decision-making. We discuss how this is achieved in the MST2/Hippo-Raf-1 signaling network with the help of mathematical modeling and model-based analysis, which showed that competing protein interactions with affinities controlled by dynamic protein modifications can function as Boolean computing devices that determine cell fate decisions. In addition, we discuss areas of interest for future research and highlight how systems approaches would be of benefit.

Compact and informative representation of functional connectivity for predictive modeling.

Resting state functional connectivity holds great potential for diagnostic prediction of neurological and psychiatric illness. This paper introduces a compact and information-rich representation of connectivity that is geared directly towards predictive modeling. Our representation does not require a priori identification of localized regions of interest, yet provides a mechanism for interpretation of classifier weights. Experiments confirm increased accuracy associated with our representation and yield interpretations consistent with known physiology.

One Hippo and many masters: differential regulation of the Hippo pathway in cancer.

The Hippo/MST2 (mammalian sterile 20-like kinase 2) pathway is a signalling cascade evolutionarily conserved in its structure. Originally described in Drosophila melanogaster as a regulator of organ size, this pathway has greater functions in mammals. Disturbance of mammalian MST2 pathway is associated with tumorigenesis by affecting apoptosis, cell cycle and polarity. In addition, this pathway has been shown to cross-talk with mitogenic pathways at multiple levels. In the present mini-review, we discuss our contribution highlighting the regulation of MST2 signalling by frequently observed oncogenic perturbations affecting mitogenic pathways. In particular, we review the role of RAS isoforms and PI3K (phosphoinositide 3-kinase)/Akt in the regulation of MST2 activity by phosphorylation. We also put the emphasis on RAF-induced control of MST2 signalling by protein-protein interactions. Finally, we recapitulate some of the direct mechanisms, such as ubiquitin-dependent degradation or gene silencing by promoter hypermethylation, involved in MST2 pathway component down-regulation in cancers.

Protein interaction switches coordinate Raf-1 and MST2/Hippo signalling.

Signal transduction requires the coordination of activities between different pathways. In mammalian cells, Raf-1 regulates the MST-LATS and MEK-ERK pathways. We found that a complex circuitry of competing protein interactions coordinates the crosstalk between the ERK and MST pathways. Combining mathematical modelling and experimental validation we show that competing protein interactions can cause steep signalling switches through phosphorylation-induced changes in binding affinities. These include Akt phosphorylation of MST2 and a feedback phosphorylation of Raf-1 Ser 259 by LATS1, which enables Raf-1 to suppress both MST2 and MEK signalling. Mutation of Raf-1 Ser 259 stimulates both pathways, simultaneously driving apoptosis and proliferation, whereas concomitant MST2 downregulation switches signalling to cell proliferation, transformation and survival. Thus, competing protein interactions provide a versatile regulatory mechanism for signal distribution through the dynamic integration of graded signals into switch-like responses.

Topological methods reveal high and low functioning neuro-phenotypes within fragile X syndrome.

Fragile X syndrome (FXS), due to mutations of the FMR1 gene, is the most common known inherited cause of developmental disability as well as the most common single-gene risk factor for autism. Our goal was to examine variation in brain structure in FXS with topological data analysis (TDA), and to assess how such variation is associated with measures of IQ and autism-related behaviors. To this end, we analyzed imaging and behavioral data from young boys (n = 52; aged 1.57-4.15 years) diagnosed with FXS. Application of topological methods to structural MRI data revealed two large subgroups within the study population. Comparison of these subgroups showed significant between-subgroup neuroanatomical differences similar to those previously reported to distinguish children with FXS from typically developing controls (e.g., enlarged caudate). In addition to neuroanatomy, the groups showed significant differences in IQ and autism severity scores. These results suggest that despite arising from a single gene mutation, FXS may encompass two biologically, and clinically separable phenotypes. In addition, these findings underscore the potential of TDA as a powerful tool in the search for biological phenotypes of neuropsychiatric disorders.

Impacts from control operations on a recreationally hunted feral swine population at a large military installation in Florida.

Feral swine were targeted for control at Avon Park Air Force Range in south-central Florida to avert damage to sensitive wetland habitats on the 40,000-ha base. We conducted a 5-year study to assess impacts from control to this population that had been recreationally hunted for many years. Control was initiated in early 2009. The feral swine population was monitored from 2008 to 2012 using a passive tracking index (PTI) during the dry and wet seasons and using recreational hunter take rates from the dry season. All three indices showed substantial feral swine declines after implementing control, with indices leveling for the final two study years. Military missions and recreational hunting seasons impacted temporal and spatial consistency of control application, thereby limiting further impacts of control efforts on the feral swine population. The PTI was also able to monitor coyotes, another invasive species on the base, and detect Florida black bear and Florida panther, species of particular concern.